PONE-D-22-06035Comparative analysis of dCas9-VP64 variants and multiplexed guide RNAs mediating CRISPR activationPLOS ONE

Dear Dr. Miner,

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PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Reviewer #1: In this study, authors analyzed multiple combinations of CRISPR/Cas9-mediated activation system. They found dCas9-VP64 and dCas9-p300 combined with MCP-VP64 is more efficient than direct fusion of VP64 to the N-terminus of dCas9 in transcriptional activation for both endogenous and exogenous genes. This study is well-defined and can provide researchers with another efficient tool for gene functional study. However, there are some points that should be revised and addressed by authors so that readers can better understand.

The major point is that the statistical comparisons are unclear and require better clarification.

- Figure 1: The statistical significance was labeled only on some bars. Does this mean only marked bars are significant compared to control? And please clarify the comparisons by drawing lines on the graphs for those were described in text or apply a different symbol as author showed in later figures. Why use two-way ANOVA in this figure?

- Figure 2: 2VP is not significant?

- Figure 3: Not significant for p300 and VP-p300 in ASCL1? p300+MV is not novel? Please double check the statistical significance of VP-p300+MV in ASCL1 and MYOD1.

- Figure4: Only single p300+MV of IL1RN is statistically significant when compared to control? In IL1RN, is multiplexing gRNAs compared to single gRNA of p300+MV that significant (***)?

- Figure 5: Check labeling for statistical comparisons. I see no description for the activity of VP+MV and p300+MV in IL1RN.

Minor points:

- Please unify the labeling by using either control or non-induced control (used in Figure 2) in figure legends.

- Author used abbreviation for all figures except Figure 6, please unify.

- Please consider combining similar figures, for example, Fig. 2 & 3, Fig. 4 & 5 and Fig. 6 & 7.

- Please consider using fluorescent intensity to convey the strength of reporter activation in Figure 6C.

Reviewer #2: The manuscript is a logical and useful extension of previous work on modified CRISPR tools. The methods used are standard and the outcomes are helpful to the research community. While not surprising, the experiment that shows the impact of multiplexing gRNAs targeting a single gene is definitely a useful to researchers.

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Comparative analysis of dCas9-VP64 variants and multiplexed guide RNAs mediating CRISPR activation

PONE-D-22-06035R1

Dear Dr. Miner,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Gaurav Varshney, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

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6. Review Comments to the Author

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Reviewer #1: (No Response)

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Reviewer #1: No

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